Competition-based ELISA discriminates between pathogenic high-affinity and nonpathogenic low-affinity anti-FVIII antibodies. High-affinity pathogenic antibodies (dark blue) interact with a low concentration of FVIII (green) in the fluid phase and therefore do not bind to FVIII insolubilized on a solid surface. On the contrary, low-affinity nonpathogenic antibodies (light blue) do not make complexes with a low concentration of FVIII in solution but bind to insolubilized FVIII, possibly because each antibody can interact with 2 FVIII molecules that are held in close proximity at the solid surface. High- and low-affinity antibodies bind to FVIII when it is present at a high concentration in solution. Only a fraction of low-affinity antibodies are then still available to interact with insolubilized FVIII. High-affinity antibodies are found only in hemophilia A patients treated with FVIII concentrates who develop FVIII inhibitors or in patients with acquired hemophilia A. The low-affinity anti-FVIII antibodies are found in hemophilia A patients with a normal response to FVIII concentrates and in healthy controls.